Cell Discovery

Stem cells proliferate rapidly to maintain fast tissue turnover during regeneration. However, the feedback mechanisms in stem cells that prevent hyperproliferation remain unclear, and their dysregulation can lead to organ failure and cancer. Here, we identified nuclear factor-Y (NF-Y) as the transcriptional repressors to maintain the stem cell quiescence during intestinal homeostasis. We found that NF-Y negatively regulates intestinal stem cell (ISC) proliferation through preferentially occupying the promoters of EGFR signaling pathway components Egfr/Mkp3/Raf/Ras/pointed, via the action of histone acetyltransferase Nejire (Nej)/p300 dependent transcription regulation. While the loss of NF-Y enhances ISC proliferation, cell death and sensitivity to stress and tumor induced mortality. Moreover, NF-Y acts together with Nej to restrict Egfr expression and suppress ISC hyperproliferation. Together, these results demonstrate NF-Y acts with Nej serve as a key negative feedback module to orchestrate transcription initiation and termination of growth signaling in the control of stem cell activity in homeostatic and disease conditions.

Multi-omics and spatial-omics technologies are exploding in use, producing increasingly complex datasets. Existing bioinformatics tools are developing rapidly but fail to fully enforce the FAIR principles, leaving the field vulnerable to escalating issues in computational reproducibility. Here, we introduce a reproducible-by-design paradigm represented in an omics data processing package, RomicsProcessor. At its core, the "Romics_object", which is a self-contained digital artifact that encapsulates the full history of the data from the original data to the fully processed state, capturing the details of the transformative steps and the required dependencies. This architecture ensures that computational workflows are fully portable and reproducible. In this manuscript, we demonstrate RomicProcessors computational capabilities and scalability on diverse datasets, including bulk proteomics, large-scale multiplexed immunofluorescence, and multi-batch mass spectrometry imaging. Providing a robust framework for truly FAIR Data Principles-based analysis, RomicsProcessor is a blueprint for the next generation of reproducible bioinformatics tools that can dramatically accelerate discovery in multi-omics biology in the era of artificial intelligence.

The gut microbiota influences colorectal cancer (CRC) progression, primarily through the secretion of small molecule metabolites. While numerous microbial products are known to drive CRC, endogenous protective mechanisms remain largely uncharacterized. Utilizing a folate metabolomics platform, we demonstrate that the healthy gut microbiota produces folinic acid (FA), a known chemotherapeutic adjuvant also known as leucovorin. This microbially derived folinic acid is progressively depleted in mouse models of colitis-associated CRC and in human clinical metagenomic cohorts with advancing disease severity. Mechanistically, folinic acid acts as a signaling molecule that directly binds and inhibits the intracellular protease calpain-2. This interaction stabilizes epithelial E-cadherin protein expression and suppresses CRC epithelial-to-mesenchymal transition driving metastasis. Genetically manipulating gut microbial production of FA is sufficient to modulate CRC in vivo, even in the presence of chronic inflammation. This study reframes folinic acid from a chemotherapeutic enhancer to an endogenous microbial metabolite that actively suppresses CRC progression.

BackgroundTuberculosis remains a leading global infectious killer, with BCG offering inconsistent adult protection and rising drug-resistant strains demanding novel vaccine strategies. We report the first multi-epitope vaccine construct simultaneously targeting three previously unexplored Mycobacterium tuberculosis virulence proteins; EccB3, MycP, and polyketide synthase which collectively govern nutrient acquisition, ESX secretion integrity, and innate immune evasion. MethodsUsing a reverse vaccinology pipeline, B-cell, CTL, and HTL epitopes were predicted, filtered for allergenicity, toxicity, and IFN-{gamma} induction, then assembled into an 823-residue chimeric construct incorporating beta-defensin and PADRE adjuvants with AAY/GPGPG linkers, covering [~]90% global HLA diversity. The construct underwent AlphaFold structure prediction, 3DRefine refinement, disulfide engineering, PROCHECK/ProSA validation, ClusPro 2.0 docking against TLR1/TLR2, and C-IMMSIM immune simulation. ResultsThe construct (82.3 kDa, instability index 32.48) showed strong structural quality (94.7% favoured Ramachandran residues), stable TLR1/TLR2 binding (weighted energy: -1,371.0 kcal/mol), and robust in silico immune responses and durable memory cell formation following booster simulation. ConclusionThis computationally validated construct represents a promising multi-target TB vaccine candidate warranting experimental advancement.

Efficient systemic delivery to the lung remains a major barrier for adeno-associated virus (AAV)-mediated pulmonary gene therapy, particularly when pre-existing immunity limits the use of conventional capsids. Here, we evaluated Bovine AAV, a phylogenetically divergent capsid, as candidate vector for lung-directed gene transfer. In adult C57BL/6J mice, intravenous delivery of Bovine AAV resulted in robust and preferential lung transduction comparable to AAV4, with predominant targeting of alveolar type I pneumocytes and pulmonary endothelial cells. In primary human lung-resident cells, Bovine AAV was particularly effective in microvascular endothelial cells, a target poorly transduced by AAV4 in vitro. Bovine AAV demonstrated scalable production with yield, purification performance, capsid quality, and genome integrity comparable to AAV9. In sera from healthy adults from the United States and Switzerland, Bovine AAV showed intermediate neutralization frequencies, lower than AAV2 and AAV4 but higher than AAV5 and AAV9. Of relevance, Bovine AAV maintained in vivo transduction efficiency in mice previously immunized with a pool of human and non-human primate-derived AAV capsids, including AAV4. Together, these results position Bovine AAV as a promising lung-tropic and immune-distinct vector for pulmonary gene therapy, with particular relevance for applications requiring systemic delivery in the presence of pre-existing immunity to conventional serotypes.

Macrophages are innate immune cells of embryonic or adult origin with tissue specific roles in homeostasis, disease surveillance, and wound repair that can be co-opted to promote tumor growth and spread1-11. An understanding of the specific roles of macrophage subsets in lung tumor initiation and progression could promote new therapeutic approaches for this deadly disease. Here, we show that KRASG12D mutations in lung epithelium drive proliferation of resident, embryonically-derived alveolar macrophages, which then promote tumor cell proliferation and protection from ferroptosis, leading to tumor progression. Using genetically engineered mouse models of mutant KRASG12D non-small cell lung cancer12,13, we found that alveolar macrophages accumulate by proliferation in response to tumor cell-secreted IL-34, recapitulating events observed in late embryonic lung development. Tumor alveolar macrophages in turn drive IGF-1-dependent tumor cell proliferation. Neutralization or deletion of IL-34 suppresses IGF-1 expression, reduces macrophage and tumor cell proliferation and inhibits tumor progression. High IL34 and IGF1 correlate with poor survival in KRASG12D/V lung adenocarcinomas and in other solid tumors, indicating that bi-directional proliferative signaling between resident macrophages and tumor cells can drive human lung tumor progression. These studies identify resident macrophage-tumor cell interactions as key interception points for lung cancer therapy.

RNA-binding protein LARP4 plays an important role in T cell activation and differentiation, but its role in B cell biology and the pathogenesis of systemic lupus erythematosus (SLE) remains unclear. This study found that LARP4 was specifically highly expressed in B cells of SLE patients and was positively correlated with disease activity. By constructing T cell-specific and B cell-specific conditional knockout mice, we found that deletion of LARP4 in B cells, but not in T cells, significantly alleviated pristane-induced and Bm12-induced lupus nephritis. Further analysis showed that LARP4 deletion selectively inhibited B cell differentiation into plasma cells, but did not affect germinal center B cell formation. Integrated transcriptomic and metabolomics analyses revealed that this effect is due to reduced phosphatidic acid synthesis and decreased mTORC1 activity caused by mitochondrial oxidative phosphorylation dysfunction. Furthermore, we used LIPEP, a LARP4 inhibitory peptide that effectively mimicked the therapeutic effects of LARP4 gene knockout in the MRL/lpr spontaneous lupus model and outperformed cyclophosphamide in reducing glomerular immune complex deposition and improving extrarenal dermatitis. These results indicates that LARP4 is a key metabolic checkpoint regulating B cell differentiation into Plasma cells and suggest that it may be a potential therapeutic target for SLE.

Background The 2022-2023 global mpox outbreak highlighted the critical need for robust genomic surveillance capabilities to track mpox virus (MPXV) evolution and transmission dynamics. Methods Building upon our established SARS-CoV-2 sequencing infrastructure, we implemented a Molecular Loop probe-based long-read sequencing approach using Pacific Biosciences Sequel II technology for comprehensive MPXV genomic surveillance across the United States (US). From August 2024 to June 2025, we generated 326 high-quality whole genome sequences from residual mpox-positive clinical specimens collected by Labcorp across all 10 US Department of Health and Human Services regions. Results Our analysis identified two samples containing clade Ib MPXV in January and June 2025 and captured shifting trends in clade IIb diversity, with 13 distinct lineages observed. We also identified multiple instances of large (~1.6-17.6kb) deletions proximal to the inverted terminal repeats in clade IIb genomes. APOBEC3 mutation analysis indicated substantial evidence of human-to-human transmission among both clades. Further, we observed significantly higher APOBEC3-associated SNPs per kilobase (P<0.001) in clade IIb genomic variable regions relative to their central conserved region. Our assay exhibited strong reproducibility across biological replicates from individual patients and accuracy was confirmed via parallel sequencing of select specimens by US Centers for Disease Control and Prevention (CDC) using metagenomic sequencing. We also demonstrated via custom simulation that our assay discriminates all known MPXV clades and lineages, including those we have not observed in the US. Conclusions Our integrated nationwide surveillance system facilitates real-time genomic tracking of outbreak evolution, with demonstrated capacity across SARS-CoV-2 and MPXV, positioning this platform for rapid deployment during future pathogen emergence.

Introduction: Recommendations from physicians and peers play a crucial role in promoting vaccination. This study evaluated differences in recommendations to others regarding four vaccines with varying efficacy (seasonal influenza, measles, human papillomavirus [HPV], and coronavirus disease 2019 [COVID-19]) between physicians and the general public and examined the impact of birth-year-based vaccination policy changes on these recommendations. Methods: This cross-sectional study was conducted in February 2026 among 492 physicians and 5,252 members of the general public in Japan. Consistency in recommendations across the four vaccines was assessed using the intraclass correlation coefficient (ICC[3,1]), and group differences were examined using a two-way mixed-design analysis of covariance. Multilevel regression discontinuity analyses were performed to evaluate the effects of birth-year-based vaccination policy. Results: Physicians showed significantly stronger recommendations to others than the general public, and their recommendation patterns generally reflected vaccine efficacy. However, physicians showed lower consistency across vaccine types than the general public (ICC[3,1]), driven primarily by heterogeneity in COVID-19 vaccine recommendations. Regression discontinuity analyses showed that birth-year-based vaccination policy, including routine vaccination opportunities, was significantly associated with recommendations to others for measles and HPV vaccines, independently of perceived benefits and risks. Conclusion: To improve vaccination coverage from a public health perspective, it is important for physicians to provide effective vaccination recommendations to the general public on a broader scale; however, it is also necessary to address the heterogeneity in vaccine-specific recommendation patterns among physicians, as observed for COVID-19. Routine vaccination opportunities may increase vaccination coverage not only through the routine vaccination program itself but also through peer effects among the general public. Vaccination policy may therefore influence vaccination coverage not only in the current generation but also in future generations. Designing vaccination policy should consider its long-term impact on future vaccination coverage as well as herd immunity.

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, yet the factors governing reaction longevity remain poorly understood despite their importance for high-throughput applications. Here, the three principal determinants of CFPS performance--DNA template design, reaction composition, and lysate genotype--were systematically optimized to extend reaction lifetime in a 384-well plate format. Different energy regeneration systems were evaluated through real-time pH monitoring and metabolomic analyses to identify the metabolic constraints limiting prolonged protein synthesis. Lysates prepared from engineered Escherichia coli BL21(DE3) strains were further examined to assess the contributions of DNA, RNA, and amino acid stabilization. Systematic optimization of amino acid, nucleoside triphosphate, polyethylene glycol, and lysate concentrations identified DNA template stability and amino acid preservation as the primary factors sustaining CFPS activity. Combining these improvements yielded reactions that remained productive for >14 h and produced 567 {+/-} 64 g mL-1 active deGFP. These findings establish practical strategies for extending CFPS lifetime and improving high-throughput cell-free platforms.

Aldehyde dehydrogenases (ALDH) are enzymes that catalyze the NAD(P)+-dependent oxidation of aldehydes into carboxylic acids, playing roles in detoxification, biosynthesis, and regulatory functions. Dysfunction of ALDH is associated with serious conditions such as alcohol intolerance, cancer, cardiovascular problems, and neurological disorders. In humans, ALDH1A1 and ALDH1A3 isoforms act as retinaldehyde dehydrogenases and are overexpressed in various cancers, where high levels are associated with increased tumor malignancy, cancer stem cell traits, and therapeutic resistance. ALDH1A3 is recognized as a promising target for anticancer therapies, with several inhibitors, mainly reversible, developed to specifically target it or the enzyme family. Since ALDH enzymes can also display esterase activity, we used this property to develop an in vitro assay specifically targeting the esterase function of ALDH1A3. A highly conserved active-site cysteine in ALDH1A3 is located at the bottom of two converging channels, which define the substrate- and cofactor-binding pockets. To target this catalytic cysteine, we screened a library of 3,200 cysteine-focused covalent fragments. This led to the identification of Z3405279217 (Z34), an acrylamide-based covalent compound that inhibits both ALDH1A1 and ALDH1A3 at sub-micromolar levels. Biochemical and biophysical tests confirmed that Z34 acts as a time-dependent, covalent, and irreversible binder to the active-site cysteine. In this work, we determined the Cryo-EM structure of the ALDH1A3-Z34 complex at 2.26 [A] resolution, confirming the covalent attachment to the catalytic cysteine of Z34. Notably, two mutually exclusive covalent binding modes were observed: one occupying the substrate-binding pocket and the other the cofactor-binding region. Z34 displayed unexpected binding modes within the active site and holds promise as a lead compound for future drug development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/738401v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@982d1forg.highwire.dtl.DTLVardef@ba86f2org.highwire.dtl.DTLVardef@1f19f2borg.highwire.dtl.DTLVardef@8e807_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Ser/Thr kinase ATM orchestrates cellular responses to DNA double-strand breaks (DSBs) and promotes DSB repair by homologous recombination. In this process, ATM is activated by DNA and the MRN (MRE11, RAD50, and NBS1) complex. Here we show that mutations of the conserved PIKK regulatory domain (PRD) within ATMs kinase domain can confer a maximally active state that no longer requires MRN/DNA. In ATM-knockout human cells, the PRD mutants display substantially higher phosphorylation of histone H2AX, KAP1, and CHK2 than wild-type ATM, with or without IR-induced DNA damage. Cryo-EM structures of two PRD mutants each revealed basal or activated conformations depending on bound ligands, suggesting that disrupting the ordered portion of the PRD results in an enzyme poised to transition to the active conformation. However, the identity of the active-site nucleotide is a key driver of the conformational switching. We speculate that this plasticity might be exploited to develop small-molecule ATM modulators for therapeutic applications.

Topoisomerase II (TOP2) resolves DNA topological constraints through a tightly regulated cycle of DNA double-strand cleavage and religation. Nearby DNA damage or chemotherapeutic agents such as etoposide block the DNA religation step, stabilizing TOP2-DNA cleavage complexes (TOP2ccs) at DNA double-strand breaks (DSBs). The SUMO E3 ligase ZATT (ZNF451) has recently emerged as a key effector of TOP2cc repair, but its mechanism of action remains poorly understood. Here, we show that ZATT is sufficient to resolve TOP2ccs independently of TDP2, TOP2 proteolysis, and canonical DSB repair pathways. Using Xenopus egg extracts and biochemical reconstitution, we find that ZATT salvages trapped TOP2 by promoting TOP2 release from its stalled cleavage complex. Structural modeling and targeted mutagenesis in Xenopus egg extracts and human cells identify a highly conserved hydrophobic pocket in the tower domain of TOP2 where the ZATT coiled-coil "hooks on" to promote TOP2cc resolution. Our findings reveal a new strategy to resolve TOP2ccs that bypasses the exposure of dangerous DNA breaks.

Various methods have detected miRNA-target interactions via immunoprecipitation of UV-crosslinked Argonaute ribonucleoprotein complexes, followed by intermolecular ligation of bound miRNAs to target strands, forming chimeric RNAs. To date, these methods have relied on conventional viral reverse transcriptases (RTs) to generate cDNAs for sequencing. However, crosslinked RNAs often retain adducts after purification, which can make them poor templates for viral RTs. Here, we adapted OTTR (Ordered Two-Template Relay) techniques to generate cDNAs from Ago2-bound RNAs. OTTR makes use of a modified retroelement-encoded RT, which is strongly processive even on templates with modifications or adducts. We show that this "OTTR-CLASH" method increases the frequency of generating chimeric RNAs compared to previous methods. We also developed an improved bioinformatic pipeline for analysis of these data, and we use this to catalog miRNA-target interactions not previously described in the literature. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/738487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13bc276org.highwire.dtl.DTLVardef@5beb41org.highwire.dtl.DTLVardef@b204e5org.highwire.dtl.DTLVardef@15f747d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Low response rates to immune checkpoint inhibitors (ICIs) in solid tumors are often driven by insufficient tumor-infiltrating CD8 T cells and immunosuppressive tumor microenvironment (TME). Although interleukin-2 (IL-2) potently expands and activates CD8 T cells, its clinical use is limited by rapid clearance, dose-limiting toxicity, and regulatory T cell (Treg) stimulation. Engineered IL-2 variants have not yet achieved meaningful clinical efficacy. Here, polymer-modified mesoporous silica nanoparticles displaying dense, unmodified wild-type IL-2 on their surface (IL2-NP) are developed, conferring proteolytic stability and tumor retention. IL2-NP enables avidity-mediated CD8 T cell binding and enhances proliferation and effector function without increased Treg binding or proliferation. Intratumoral IL2-NP expands CD8 T cells, increases CD8/Treg ratios, and reprograms TME through dendritic cell activation and M1-like macrophage polarization. IL2-NP induces regression of both treated and untreated distant colorectal tumors in a CD8 T cell-dependent manner. IL2-NP synergizes with ICIs and leads to complete tumor regression and immunological memory that protect against rechallenge. Treatment is well tolerated, with strong efficacy also observed in triple-negative breast and metastatic ovarian cancer models. Overall, intratumoral IL2-NP elicits robust systemic antitumor immunity, offering a promising strategy to enhance ICIs, cancer vaccines, and adoptive T-cell therapies. Graphical abstractThis work introduces a nanoparticle platform that overcomes major shortcomings of IL-2 immunotherapy by presenting wild-type IL-2 at high density on the nanoparticle surface, thereby increasing binding avidity to effector T cells. The resulting IL-2 nanoparticles enhance cytotoxic T cell expansion, reprogram the tumor microenvironment, and augment responses to immune checkpoint blockade to achieve robust ant-tumor immune response in mouse tumor models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/738558v1_ufig1.gif" ALT="Figure 1"> View larger version (82K): org.highwire.dtl.DTLVardef@12f8c8corg.highwire.dtl.DTLVardef@b46b1forg.highwire.dtl.DTLVardef@e4efc5org.highwire.dtl.DTLVardef@3993e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cells must be primed to rapidly induce inflammatory gene expression upon infection while also tuning the level of induction to avoid immunopathology. Here, we identify RNA polymerase III (Pol III), best known for transcribing noncoding RNAs, as a dual-function regulator of RNA polymerase II (Pol II)-dependent inflammatory gene expression. Pol III is selectively enriched at promoters of innate immune, pro-inflammatory, and stress-response genes, where it maintains chromatin accessibility and supports basal transcription. Upon infection with murine gammaher-pesvirus 68 (MHV68), Pol III redistributes from these promoters to retrotransposon loci, coinciding with enhanced expression of inflammatory genes. Depletion of the Pol III transcription factor Brf1 further amplifies inflammatory transcription during infection with MHV68, herpes simplex virus-1, and influenza A virus. Genes restrained by Pol III have TATA-box-enriched promoters and are functionally dependent on TATA-binding protein (TBP), suggesting that Pol III modulates inflammatory gene expression by competing with Pol II for shared transcriptional machinery. Thus, Pol III is a chromatin licensor in uninfected cells and an inflammation rheostat during viral infection. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/738346v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d99325org.highwire.dtl.DTLVardef@4b781dorg.highwire.dtl.DTLVardef@bad1b6org.highwire.dtl.DTLVardef@11e41a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

"Mirror life", self-replicating organisms composed of non-natural-chirality biomacromolecules, presents a future threat with potentially global consequences. Consequently, there is strong agreement among experts that it should not be created. However, there is some disagreement over how effective existing medical countermeasures might prove against mirror bacteria in the event that they were created. Here, we leverage computational chemistry methods including docking and molecular dynamics to determine the likely binding efficacy of existing antibiotics against natural and mirror bacterial protein targets. We find that most existing antibiotics fail to bind to mirror bacterial protein targets, unlike their natural-chirality targets. This suggests altered binding of current medical countermeasures, which may impact the antimicrobial activity against mirror bacteria were the latter were created.

Efferocytosis, the recognition, engulfment, and degradation of apoptotic cells by phagocytes, is essential for tissue homeostasis and development, and its failure contributes to chronic inflammation and neurodegeneration. The amyloid precursor protein (APP), a central pathogenic factor in Alzheimers disease, retains physiological functions independent of amyloid production that remain poorly understood. Here, we identify a conserved, non-amyloidogenic role for APP in regulating apoptotic cell degradation via the endolysosomal pathway. Using Drosophila APPL as a model, structure-function analysis demonstrated that the intracellular internalization domain of APPL, but not its secreted ectodomain, is required for efficient apoptotic cell degradation. Immunoprecipitation coupled with mass spectrometry revealed a physical interaction between APPL and the microtubule severing ATPase Spastin, mediated by the microtubule-interacting and trafficking domain of Spastin. APPL interacts with Spastin on endosomal microtubules and modulates the dynamics of the Spastin-ESCRT-III complex, enabling Spastin to sever microtubules and promote endosomal tubule fission. Loss of APPL disrupts this process, causing aberrant endosomal tubulation and impaired lysosome biogenesis. Furthermore, it compromises the function of residual lysosomes, characterized by reduced acidity, diminished proteolytic activity, and increased lysosomal damage, which ultimately impairs the degradation of engulfed apoptotic cells. Critically, this phenotype is evolutionarily conserved in C. elegans and mice. Together, these findings establish a conserved APP-Spastin axis that regulates endolysosomal homeostasis and apoptotic cargo digestion. This reveals a critical non-amyloidogenic function of APP in maintaining tissue homeostasis through efficient efferocytosis, with broad implications for inflammatory and neurodegenerative disorders that warrant further investigation.

Background and Aims The optimal treatment for first episodes and first recurrences of Clostridioides difficile infections (CDI) is unknown and there is emerging evidence for pulse and taper (P-T) regimens. Therefore, we sought to estimate the relative efficacy of treatment options. Methods MEDLINE and CENTRAL were searched from database inception to May 21, 2025 and unpublished conference abstracts were searched from recent infectious disease conferences. RCTs on the treatment of first episodes or first recurrences of CDI comparing fixed-dose or P-T regimens of fidaxomicin or vancomycin were included. The primary and secondary outcomes were 40- and 56-day CDI recurrence, respectively. A random-effects network meta-analysis on the risk ratio (RR) scale was conducted using a standard regimen (10-14 days) of vancomycin as the comparator. Treatments were ranked using the surface under the cumulative ranking curve (SUCRA). Results 8 RCTs were included comprising a total of 2181 patients. For 40-day recurrence, fidaxomicin P-T had the highest probability of ranking best (RR=0.10, 95%Confidence Interval [95%CI]=0.10-0.49, SUCRA=1.00), followed by vancomycin P-T (RR=0.49, 95%CI=0.32-0.76, SUCRA=0.61), fixed-dose fidaxomicin (RR=0.61, 95%CI=0.49-0.76, SUCRA=0.39), and, finally, fixed-dose of vancomycin (SUCRA=0.00). The treatments ranked in the same order for 56-day recurrence, though only 3 RCTs reported on this timepoint. Conclusion Vancomycin P-T, fidaxomicin P-T, and fixed-dose fidaxomicin were all superior to a fixed-dose vancomycin. Head-to-head comparative effectiveness RCTs are needed to quantify their relative effect sizes of and impact on long-term prevention of recurrent CDI.

Co-transcriptional splicing is a conserved feature of eukaryotic gene expression. However, establishing the functional nature of this process has been difficult. Here using high throughput CRISPR/Cas9 screens we surprisingly find that SF3B3, the third largest subunit of the U2 snRNP complex, is a major regulator of RNA Pol II pause release and processivity. Remarkably, the absence of SF3B3 dramatically perturbs transcription but U2 snRNP assembly and RNA splicing remains unaffected. Mechanistically, SF3B3 coordinates the chromatin occupancy of transcriptional kinases (CDK9/12/13) alongside the PAF1c and Integrator complexes to regulate Pol II. Structure / function analyses of SF3B3 revealed that a metazoan specific 18aa sequence within its disordered tail phenocopies its loss and mediates the physical association and stability of SF3B5. We show that loss of SF3B5 mirrors SF3B3 deficiency suggesting this submodule, although resident within the U2 snRNP complex, evolved to primarily coordinate RNA Pol II in a splicing-independent manner.